System, apparatus and method for material preparation and/or handling

ABSTRACT

Oscillating angularly rotating a container containing a material may cause the material to be separate. Denser or heavier material may unexpectedly tend to collected relatively close to the axis of rotation, while less dense or light material may tend to collect relatively away from the axis of rotation. Oscillation along an arcuate path provides high lysing efficiency. Alternatively, a micromotor may drive an impeller removably received in a container. Lysing may be implemented in batch mode, flow-through stop or semi-batch mode, or flow-through continuous mode. Lysing particulate material may exceed material to be lysed or lysed material and/or air may be essentially eliminated from a chamber to increase lysing efficiency.

TECHNICAL FIELD

The present disclosure relates to the separation of matter, for exampleparticles or other material in a suspension. The present disclosure alsorelates to lysing and in particular to systems, apparatus and methods toperform lysing of a material to be lysed using a lysing particulatematerial.

BACKGROUND

There are numerous applications that require the separation of material,for example particulate or other matter in suspension. One commonapproach is to employ a centrifuge to separate relatively heaviermaterial from relatively light material. Centrifuges typical include acontainer to hold the material, a drive system including a motor andtransmission or linkage coupled to rotate the container about a fixedaxis of rotation. The material in the container separates based ondensity under centripetal acceleration, with denser or heavier materialtending to collect at a perimeter relatively away from the axis ofrotation and with the less dense or lighter material tending to collectrelatively closer to the axis of rotation.

Centrifuges may be used on a large variety of material fromparticulates, to fluids, to gases, and combinations of the same.Centrifuges are often used to separate biological material, for examplein preparing samples for analysis of the composition of specificbiological materials, such as proteins, lipids, and nucleic acids eitherindividually or as complexes. A centrifuge may be used to isolatecertain organelles-nuclei, mitochondria, lysosomes, chloroplasts, and/orendoplasmic reticulum.

Lysis of biological material, for example cell lysis, is used to analyzethe composition of specific biological materials, for example proteins,lipids, and nucleic acids either individually or as complexes. If a cellmembrane is lysed then certain organelles-nuclei, mitochondria,lysosmes, chloroplasts, and/or endoplasmic reticulum may be isolated.Such may be analyzed using PCR, electron microscopy, Western blotting orother analysis techniques.

There are numerous approaches to performing lysis. For example,enzymatic approaches may be employed to remove cell walls usingappropriate enzymes, in preparation to cell disruption or to prepareprotoplasts. Another approach employs detergents to physically disruptcell membranes. These chemical approaches may adversely affect theresulting product, for example degrading the bio-products beingreleased. Consequently, chemical approaches may, in some instances, notbe practical.

Yet another approach employs ultrasound to produce cavitation andimpaction for disrupting the cells. Such an approach may not achieve ashigh a lysis efficiency as may be required or desired for manyapplications.

Yet still another approach employs beads (e.g., glass or ceramic) whichare agitated, for example, via a vortex mixer. Such an approachsuccessfully addresses the issues raised by chemical lysis approaches,yet improvements in such an approach are desirable.

BRIEF SUMMARY

There is a need for other approaches to separating materials. Suchapproaches may provide quicker separation, more thorough separation, ormay separate materials in a different manner than previous approaches.

There is also a need for bead-based lysing apparatus and methods thatare more efficient than current lysing apparatus. Such may reduce theamount of time required to process a sample (i.e., material to be lysed)and/or increase throughput. Such may also increase the level orthoroughness of lysing, producing greater amounts of lysed material froma given sample size. There is also a need for lysing apparatus andmethods that operate on sample sizes that are relatively small (e.g., 10micro-liters) compared to conventional lysing apparatus. Such may enablelysing to be performed where a relatively small amount of a sample isavailable and/or reduce costs. Such may also reduce the amount of lysingparticulate material that is required, also providing cost reductions.Such may also allow higher frequency oscillation, thereby increasingefficiency, while maintaining reasonable lifetime or fatiguecharacteristics. There is also a need to efficiently and reliably lysetypically difficult to lyse material, for example spores. There is afurther need for the ability to perform flow-through lysing. Such mayallow large quantities of small samples to be processed over time, forexample processing small samples taken every minute over a long periodof time (e.g., day, week, month, and/or years). There is also a need forlysing equipment that is small and hence portable, and that isrelatively inexpensive yet sufficiently robust to withstand travel orharsh operating environments.

A system to perform lysis on material to be lysed may be summarized asincluding an arm having an attachment location to at least temporarilyattach a container that at least temporarily holds a material to belysed and a particulate lysing material; a motor operable to provide adrive force; and a drive mechanism coupled to transfer the drive forceof the motor into oscillation of the attachment location of the armalong an arcuate path. The arm may be a rigid arm that does not flexunder a load in response to the oscillation of the attachment locationof the arm along the arcuate path. The arm may be a flexible arm thatflexes under a load in response to the oscillation of the attachmentlocation of the arm along the arcuate path.

The system to perform lysis may further include a holder at theattachment location, the holder configured to removably hold thecontainer. The system may include the container and the particulatelysing material. In some embodiments the container is non-removablyfixed to the arm at least proximate the attachment location. Thecontainer may have a first opening and at least a second opening spacedfrom the first opening, the first and the second openings to providefluid communication into the chamber from an exterior thereof. Thecontainer may include a first filter positioned in the chamber and asecond filter positioned in the chamber spaced from the first filter toform a particulate retainment area therebetween, the particulateretainment area positioned between the first and the second openings,the first and the second filters each having a plurality of aperturessized to substantially pass the material to be lysed and to block theparticulate material. The first filter, the second filter and theparticulate material may form a cartridge that is selectivelyreplaceable in the chamber. The plurality of beads may include at leastone of ceramic beads, glass beads, zirconium beads, metal beads, plasticbeads, and sand and wherein the plurality of beads have diameters in therange of approximately 10 microns to approximately 600 microns. When inuse a volume of the particulate matter may be greater than a volume ofmaterial to be lysed. When in use there may essentially be no air in thechamber.

The system may further include a pump operable to pump the material tobe lysed through the chamber. The pump may be configured tointermittently pump the material to be lysed through the chamber. Thematerial to be lysed may have a residence time in the chamber that maybe sufficient to achieve a defined level of lysing. The pump maycontinuously pump the material to be lysed through the chamber. Given alength of the chamber, a flow rate of the pump may be such that thematerial to be lysed spends sufficient time (i.e., desired or definedresidence time) in traversing the chamber from the first opening to thesecond opening to achieve a defined level of lysing.

The system may further include a first tube coupled to provide fluidcommunication to the first opening for the material to be lysed to thefirst opening; and a second tube coupled to provide fluid communicationfrom the second opening for a material that has been lysed. Ends of atleast one of the first and the second tubes may be reinforced. Ends ofat least one of the first and the second tubes may be reinforced withadditional tubes that are concentric about the ends of the tube. Alength of at least one of the first and the second tubes may be suchthat the length does not restrict the oscillation of the attachmentlocation. The length of at least one of the first and the second tubesmay be such that the at least one of the first and the second tubes doesnot resonate in response to the oscillation of the attachment locationof the arm along an arcuate path. A respective length of each of thefirst and the second tubes may be sufficiently long so as to notrestrict the oscillation of the attachment location and are sufficientlyshort such that the first and the second tubes do not resonate duringuse.

The drive mechanism may consist of a four-bar linkage including a firstbar rotationally driven by a motor, the bar connected by a hinge to asecond bar that serves as a connecting rod. A third and a fourth barsboth pivot about a central fixed axis with a fixed angle between them.The end of the second bar that serves as the connecting rod is connectedby a hinge to the third bar whose length determines the angle ofrotation of the third and the fourth bars. The length of the fourth baris the radius of curvature of the arcuate motion of the lysis chamber,which is coupled or connected to the fourth bar.

A method of lysing a material to be lysed may be summarized as includingreceiving a material to be lysed in a chamber that contains aparticulate lysing material; oscillating the chamber containing thematerial to be lysed and a particulate lysing material along an arcuatepath to produce a lysed material; and removing the lysed material fromthe chamber.

The method of lysing a material to be lysed may further include pumpingthe material to be lysed into the chamber.

The method may further include intermittently pumping the material to belysed into the chamber while oscillating the chamber. Intermittentlypumping the material to be lysed into the chamber while oscillating thechamber may include intermittently pumping the material to be lysed intothe chamber such that the material to be lysed spends sufficient time inthe chamber to achieve a desired level of lysing. Intermittently pumpingthe material to be lysed into the chamber while oscillating the chambermay include intermittently pumping the material to be lysed into thechamber such that the chamber is completely evacuated of the lysedmaterial during each cycle of the intermittent pumping. The chamber maybe completely evacuated of the lysed material during each cycle of theintermittent pumping by the pumping into the chamber of more material tobe lysed.

The method may further include continuously pumping the material to belysed into the chamber while oscillating the chamber. The method mayfurther include adjusting a flow rate of the pumping of the material tobe lysed into the chamber based on a length of the chamber, a flow rateof the pump is such that the material to be lysed spends sufficient timein the chamber (i.e., residence time) to achieve a desired level oflysing.

The method may further include directing the lysed material removed fromthe chamber to at least one analysis device. The method may furtherinclude evacuating the chamber with an inert fluid.

A method of lysing a material to be lysed may be summarized as includingreceiving a first cartridge having a chamber that contains a particulatelysing material and a material to be lysed; and oscillating the firstcartridge having the chamber that contains the material to be lysed andthe particulate lysing material along an arcuate path to produce a lysedmaterial.

The method may further include receiving a second cartridge in place ofthe first cartridge, the second cartridge having a chamber that containsa particulate lysing material and a material to be lysed; andoscillating the second cartridge having the chamber that contains thematerial to be lysed and the particulate lysing material along anarcuate path to produce a lysed material. Receiving a first cartridgemay include receiving the first cartridge in a mounting bracket at anattachment point of an arm. Oscillating the first cartridge may includeoscillating a rigid arm on which the first cartridge is mounted.Oscillating the first cartridge may include oscillating a flexible armon which the first cartridge is mounted.

An article to perform flow-through lysis on material to be lysed may besummarized as including at least one wall forming at least one chamberhaving a first opening and at least a second opening spaced from thefirst opening, the first and the second openings to provide fluidcommunication into the chamber from an exterior thereof; a particulatelysing material received in the chamber, the particulate materialincluding a plurality of particles sized to lyse a material to be lysed;a first filter received in the chamber between the first opening and theparticulate material, the first filter having a plurality of aperturessized to substantially pass the material to be lysed and to retain theparticulate material; and a second filter received in the chamberbetween the second opening and the particulate material, the secondfilter having a plurality of apertures sized to pass the material to belysed and to retain the particulate material, wherein the first filterand the second filter form a particulate retainment area therebetween.

The article may further include an attachment structure proximate thefirst opening. The article may further include a first attachmentstructure to attach a first tube to the first opening; and a secondattachment structure to attach a second tube to the second opening.

The article may further include a first nipple to attach a first tubeabout the first opening; and a second nipple to attach a second tubeabout the second opening. The at least one wall may be elongated andhave a first end and a second end opposed to the first end. The firstopening may be at the first end and the second opening may be at thesecond end. At least one wall may be cylindrically tubular.

The particulate material may be a plurality of beads. The plurality ofbeads may include at least one of ceramic beads, glass beads, zirconiumbeads, metal beads, plastic beads, and sand. The plurality of beads mayhave diameters in the range of approximately 100 microns. The pluralityof beads may have diameters in the range of 50 microns to 150 microns.When in use, a volume of the particulate matter may be greater than avolume of material to be lysed. When in use there may be essentially noair in the chamber. The chamber may have a volume that holds less than60 μl of the material to be lysed. The chamber may have a volume thatholds approximately 10 μl to approximately 40 μl of the material to belysed. The first and the second filters may be fixed to the wall.

A system to perform lysis may be summarized as including a containerhaving at least one chamber to hold a material to be lysed and a lysingparticulate material, the chamber having a first opening and at least asecond opening to provide fluid communication into the chamber from anexterior thereof; an impeller having a number of blades received in thechamber of the container; and a micromotor coupled to turn the impeller.The first opening may provide an entrance for material to be lysed andthe second opening may provide an exit for material that has been lysed.

The chamber may have a third opening, at least a portion of themicromotor may be received by the third opening and may seal the thirdopening. The micromotor may be removably received in the first thirdopening. The micromotor may be disposable.

The container may further include at least a first filter positionedbefore the exit in a flow path, the first filter having a plurality ofapertures sized to substantially pass material that has been lysed andto substantially block lysing material. The container may furtherinclude at least a second filter positioned following the entrance inthe flow path, the second filter having a plurality of apertures sizedto substantially pass material to be lysed and to substantially blocklysing material.

The micromotor may pulsate. The micromotor may drive the impeller at arate of greater than 10,000 RPM in the presence of liquid and beads. Themicromotor may drive the impeller at a rate of approximately 50,000 RPM,when not in the presence of liquid and beads.

A method of system to perform lysis, may be summarized as includingreceiving a material to be lysed via an entrance in at least one chamberof a container that holds a lysing particulate material; driving animpeller having a number of blades received in the chamber of thecontainer via a micromotor; and expelling a material that has been lysedvia an exit from the chamber of the container.

Expelling a material that has been lysed via an exit may includeexpelling the material that has been lysed via a first filter positionedbefore the exit in a flow path, the first filter having a plurality ofapertures sized to substantially pass the material that has been lysedand to substantially block the lysing particulate material. Receiving amaterial to be lysed via an entrance may include receiving the materialto be lysed via a second filter positioned following the entrance in theflow path, the second filter having a plurality of apertures sized tosubstantially pass the material to be lysed and to substantially blocklysing particulate material.

The method of system to perform lysis may further include intermittentlypumping the material to be lysed into the at least one chamber via theentrance. The method may further include continuously pumping thematerial to be lysed into the at least one chamber via the entrance.

Driving an impeller may include pulsating the impeller. Driving animpeller may include driving the impeller at a rate of greater than10,000 RPM in the presence of liquid and beads. The method may furtherinclude replacing the micromotor with a new micromotor. The method mayfurther include disposing the micromotor.

A system to perform lysis, may be summarized as including a firstcontainer having at least one chamber to hold a material to be lysed anda lysing particulate material, the chamber having a single opening toprovide fluid communication into the chamber from an exterior thereof;an impeller having a number of blades received in the chamber of thefirst container; and a micromotor coupled to turn the impeller, at leasta portion of the micromotor removably received in the single opening ofthe first container to seal the single opening in use. The micromotormay be disposable. The micromotor may be removably received by a singleopening of a second container after removal from the single opening ofthe first container. The micromotor may pulsate. The micromotor maydrive the impeller at a rate of greater than 10,000 RPM in the presenceof liquid and beads.

A method of operating a system to perform lysis may be summarized asincluding receiving a material to be lysed via an entrance in at leastone chamber of a first container that holds a lysing particulatematerial; locating an impeller in the chamber of the first container viathe entrance; closing the entrance of the first container with amicromotor that is coupled to drive the impeller; and driving theimpeller to circulate the material to be lysed and the lysingparticulate material in the chamber of the first container.

The method of system to perform lysis may further include removing themicromotor from the entrance of the first container and removing amaterial that has been lysed via the entrance of the first container.Removing a material that has been lysed via the entrance of the firstcontainer may include withdrawing the material that has been lysed usinga pipette. Driving the impeller may include pulsating the impeller. Themethod may further include reusing the micromotor with a secondcontainer. The method may further include disposing of the micromotor.

A system to separate materials may be summarized as including: a base;an actuator coupled to the base and selectively operable to provide adrive force; and a drive mechanism coupled to the base and coupled totransfer the drive force of the motor into a high frequency oscillatoryangular rotation of a container about an axis of rotation. The actuatormay be an electric motor.

The system may further include a holder coupled to the drive mechanismfor movement thereby, the holder configured to removably hold thecontainer.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated and the container isnon-removably fixed to the drive mechanism.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated and at least one innerport to provide fluid communication between the interior of thecontainer and an exterior thereof, the at least one inner portpositioned relatively proximate the axis of rotation with respect to anarc defined by an oscillatory movement of an outer most portion of thecontainer from the axis of rotation.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated and at least one innerport to provide fluid communication between the interior of thecontainer and an exterior thereof, the at least one inner portpositioned at an inner periphery of the container.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated and at least one outerport to provide fluid communication between the interior of thecontainer and an exterior thereof, the at least one outer portpositioned relatively distal from the axis of rotation.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated and at least one outerport to provide fluid communication between the interior of thecontainer and an exterior thereof, the at least one outer portpositioned at an outer periphery of the container.

The system may further include the container, wherein the container hasan interior to hold the materials to be separated, at least one innerport to provide fluid communication between the interior of thecontainer and an exterior thereof and at least one outer port to providefluid communication between the interior of the container and theexterior thereof, the at least one inner port spaced relatively closerto the axis of rotation with respect to the at least one outer port. Thecontainer may include at least one filter proximate one of the inner orthe outer ports. The at least one filter may be selectively replaceablein the container.

The system may further include a pump to pump the material to beseparated through the container. The pump may be configured tointermittently pump the material through the container. The axis ofrotation may pass through the container. The container may be spacedfrom the axis of rotation. The drive mechanism may include a four-barlinkage that may include a first member, a second member, a third memberand a fourth member, the second member coupled to the first member, thefirst member rotationally driven by a motor to eccentrically drive afirst end of the second member in a circular motion, the third barmember pivotally coupled to a second end of the second member, the thirdmember connected to the fourth member at a pivot point; where anamplitude of motion of the second member and a length of the thirdmember define a angle of motion of the third and the fourth members anda length of the fourth member defines a distance of arcuate motion. Thesystem may include a controller coupled to control a frequency of theoscillatory angular rotation of the container and selectively operableto set the frequency to a sufficiently low frequency as to cause therelatively denser material to collect relatively farther from the axisof rotation than the relatively less dense material.

A method to separate materials may be summarized as including: receivinga material to be separated in a container; oscillating angularlyrotating the container at a high frequency; and removing at least someof the separated material from the container.

The method may further include pumping the material to be separated intothe container. The method may further include intermittently pumping thematerial to be separated into the container while oscillating thecontainer. The method may further include directing at least some of theseparated material removed from the container to at least one analysisdevice. The method may further include evacuating the container with aninert fluid. The method may further include varying a speed of theoscillating angular rotating to change a direction in which particles inthe material move during separation.

Such apparatus and methods may produce unexpected results. For example,in contrast to standard centrifuges, such apparatus and methods maycause denser or heavier materials to collected relatively close to anaxis of rotation while less dense or lighter materials collectrelatively away from the axis of rotation. Additionally oralternatively, such apparatus and method may allow a direction (inwardor outward with respect to the axis of rotation) of materialaccumulation to be selected by varying a speed of the apparatus. Suchapparatus and methods may even be used to combine separate materials.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, identical reference numbers identify similar elementsor acts. The sizes and relative positions of elements in the drawingsare not necessarily drawn to scale. For example, the shapes of variouselements and angles are not drawn to scale, and some of these elementsare arbitrarily enlarged and positioned to improve drawing legibility.Further, the particular shapes of the elements as drawn are not intendedto convey any information regarding the actual shape of the particularelements, and have been solely selected for ease of recognition in thedrawings.

FIG. 1A is a front elevational view of an apparatus to perform materialseparation and/or lysis, according to one illustrated embodiment.

FIG. 1B is a front, right side, top isometric view of the apparatus ofFIG. 1A.

FIG. 1C is a front, left side, bottom isometric view of the apparatus ofFIG. 1A.

FIG. 2A is a front elevational view of the apparatus of FIG. 1A with afront cover removed, according to one illustrated embodiment.

FIG. 2B is a front, right side, top isometric view of the apparatus ofFIG. 2A.

FIG. 2C is a front, right side, bottom isometric view of the apparatusof FIG. 2A.

FIG. 3 is a front, right side isometric view of a motor and drivemechanism of the apparatus of FIGS. 1A-2C.

FIG. 4 is a schematic view of a system to perform flow-throughprocessing, including an apparatus to perform material separation and/orlysis, an upstream subsystem to provide material to be separated and/orlysed, a downstream subsystem to analyze material that has beenseparated and/or lysed, and a control subsystem, according to oneillustrated embodiment.

FIG. 5 is a cross-sectional view of a container having a chamber thathouses material to be lysed, particulate lysing material, and materialthat has been lysed, according to one illustrated embodimentparticularly useful in flow-through lysing.

FIG. 6 is a flow diagram of a method of operating an apparatus, such asthe apparatus of FIGS. 1A-4, to perform lysing.

FIG. 7 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4 according to oneembodiment.

FIG. 8 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according toanother illustrated embodiment.

FIG. 9 is a flow diagram of a method of pumping material to be lysed ina flow through lysing system such as that of FIG. 4, according to yetanother illustrated embodiment.

FIG. 10 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according to stillanother illustrated embodiment.

FIG. 11 is a flow diagram of a method of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to oneillustrated embodiment.

FIG. 12 is a flow diagram of a method of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

FIG. 13 is a method of pumping material to be lysed in a flow-throughlysing system such as that of FIG. 4, according to a further illustratedembodiment.

FIG. 14 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according to stilla further illustrated embodiment.

FIG. 15 is a method of operating a flow-through lysing system such asthat of FIG. 4 to analyze lysed material, according to one illustratedembodiment.

FIG. 16 is an exploded isometric view of a lysing apparatus according toanother illustrated embodiment.

FIG. 17 is a schematic diagram of a lysing system including a lysingapparatus, an upstream subsystem to provide material to be lysed, adownstream subsystem to analyze material that has been lysed, and acontrol subsystem, according to another illustrated embodiment.

FIG. 18 is a front elevation view of a lysing apparatus and pipetteaccording to one illustrated embodiment.

FIG. 19 shows a flow diagram of a method of operating a lysing apparatussuch as that of FIGS. 16 and 17, according to one illustratedembodiment.

FIG. 20 is a flow diagram of a method of evacuating material that hasbeen lysed from a chamber in operating a lysing apparatus such as thatof FIGS. 16 and 17, according to another illustrated embodiment.

FIG. 21 is a flow diagram of a method of receiving material to be lysedin a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to one illustrated embodiment.

FIG. 22 is a flow diagram of a method of pumping material to be lysedinto a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to one illustrated embodiment.

FIG. 23 is a flow diagram of a method of pumping material to be lysedinto a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to another illustrated embodiment.

FIG. 24 is a flow diagram of a method of operating an impeller of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 25 is a flow diagram of a method of operating an impeller of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 26 is a flow diagram of a method of replacing a micromotor of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 27 is a flow diagram of a method of operating a lysing apparatussuch as that of FIG. 18, according to one illustrated embodiment.

FIG. 28 is a flow diagram of a method of operating a lysing apparatussuch as that of FIG. 18, according to one illustrated embodiment.

FIG. 29 is a flow diagram of a method withdrawing lysed material from achamber of a lysing apparatus such as that of FIG. 18, according to oneillustrated embodiment.

FIG. 30 is a flow diagram of a method of reusing a micromotor of alysing apparatus such as that of FIG. 18, according to anotherillustrated embodiment.

FIG. 31 is a graph showing data representing an efficiency of lysis as afunction of lysing duration using an apparatus similar to that of FIG.4.

FIG. 32 is a graph showing a dependency of lysis efficiency on frequencyof oscillation.

FIG. 33 is a graph showing spore lysis as a function of lysis durationfor an apparatus similar to that of the embodiment of FIG. 16.

FIG. 34 is an isometric view of a material separation apparatusaccording to another illustrated embodiment.

FIG. 35A is a top plan view of a container to hold material to beseparated, according to one illustrated embodiment.

FIG. 35B is a side-elevational view of the container of FIG. 6A.

FIG. 36A is a top plan view of a container to hold material to beseparated, according to another illustrated embodiment.

FIG. 36B is a side-elevational view of the container of FIG. 7A.

FIG. 37A is a top plan view of a container to hold material to beseparated, according to another illustrated embodiment.

FIG. 37B is a side-elevational view of the container of FIG. 8A.

FIG. 38A is a top plan view of a container to hold material to beseparated, according to another illustrated embodiment.

FIG. 38B is a side-elevational view of the container of FIG. 9A.

FIG. 39A is a top plan view of a container to hold material to beseparated, according to another illustrated embodiment.

FIG. 39B is a side-elevational view of the container of FIG. 10A.

FIG. 40A is a top plan view of a container to hold material to beseparated, according to another illustrated embodiment.

FIG. 40B is a side-elevational view of the container of FIG. 11A.

FIG. 41 is a flow diagram of a method of operating a system to separatematerials, according to one illustrated embodiment.

FIG. 42 is a flow diagram of a method of operating a system to separatematerials, according to another illustrated embodiment.

FIG. 43 is a flow diagram of a method of operating a system to separatematerials, according to another illustrated embodiment.

FIG. 44 is a flow diagram of a method of operating a system to separatematerials, according to another illustrated embodiment.

FIG. 45 is a flow diagram of a method of operating a system to separatematerials, according to another illustrated embodiment.

FIG. 46 is a graph showing bead trajectory, linear oscillations.

FIG. 47 is a graph showing constant distance b/w neighboring beads.

FIG. 48 is a graph showing how particles that are denser or heavier thatthe fluid may move toward the rotational axis rather than moving away aswould have been expected.

FIG. 49 is a graph showing an effect of a larger Stokes number, hencesmaller drag.

FIG. 50 is a graph showing a convergence of neighboring beads.

FIG. 51A is a plan view of a lysing apparatus having Luer-Lock couplers,according to one illustrated embodiment, and two syringes coupleable tothe lysing apparatus via the couplers.

FIG. 51B is an isometric view of the lysing apparatus of FIG. 51A.

FIG. 52 is a plan view of a plurality of lysing apparatus coupledsequentially to one another, according to one illustrated embodiment.

FIG. 53A is an isometric view of a manifold or array of lysingapparatus, according to one illustrated embodiment.

FIG. 53B is an isometric view of the manifold or array of lysingapparatus carried by a frame, according to one illustrated embodiment,the lysing apparatus positioned to deposit lysed material intorespective wells of a plate.

FIG. 54A is an isometric view of a cartridge style container for use inflow through lysing showing an end cap removed from a body of thecartridge style container, according to one illustrated embodiment.

FIG. 54B is an isometric view of the cartridge style container of FIG.54A with the end cap secured to a body of the cartridge style container.

FIG. 55A is a side elevational view of a stopcock style lysing device,according to one illustrated embodiment, showing an inner portionrotated or configured to provide a first flow path via two selectedports.

FIG. 55B is a side elevational view of the stopcock style lysing deviceof FIG. 55A, showing the inner portion rotated or configured to providea second flow path via two selected ports.

FIG. 56A is an exploded isometric view of a stopcock style lysingdevice, according to one illustrated embodiment, showing an inner vesselhaving an open bottom portion, the inner vessel in a first orientationwith respect to an outer vessel.

FIG. 56B is an isometric view of a stopcock style lysing device of FIG.56A, showing an inner vessel received in an outer vessel, and an drivedevice including a motor and impeller received in the inner vessel.

FIG. 56C is an isometric view of the inner vessel of FIG. 56A, showingthe inner vessel in a second orientation, different from the orientationillustrated in FIG. 56A.

FIG. 57A is an exploded side elevational view of a stopcock style lysingdevice, according to another illustrated embodiment, showing an innervessel with a closed bottom portion.

FIG. 57B is an bottom plan view of a stopcock style lysing device ofFIG. 57A.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various disclosedembodiments. However, one skilled in the relevant art will recognizethat embodiments may be practiced without one or more of these specificdetails, or with other methods, components, materials, etc. In otherinstances, well-known structures associated with micromotors,controllers including motor controllers, and control systems such asprogrammed general purpose computing systems and the like have not beenshown or described in detail to avoid unnecessarily obscuringdescriptions of the embodiments.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to.”

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. It should also be noted that the term “or”is generally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

The headings and Abstract of the Disclosure provided herein are forconvenience only and do not interpret the scope or meaning of theembodiments.

A number of embodiments of apparatus and systems to separate materialsare described herein. The material separation apparatus and systemsperform separation on a material to be separated, for example aparticulate material in suspension, to produce separated material ormaterial that has been separated. The material to be separated may takethe form of biological materials, for example cells, spores, tissue,yeast, fungi, plants, bacteria, etc., typically suspended in a liquidmedium. For instance the material may take the forms oforganelles-nuclei, mitochondria, lysosomes, chloroplasts, endoplasmicreticulum, etc. The material may include lysing particulate material,for instance beads.

A number of embodiments of lysis apparatus and systems are describedherein. The lysis apparatus and systems perform lysis on a material tobe lysed using lysing particulate material, to produce lysed material ormaterial that has been lysed. The material to be lysed may take the formof biological materials, for example cells, spores, tissue, yeast,fungi, plants, bacteria, etc., typically suspended in a liquid medium.The lysing particulate material may take a variety of forms. Whilegenerally referred to herein as beads, the term bead is not meant to belimiting with respect to size or shape. The beads may, for example, takethe form of ceramic beads, glass beads, zirconium beads,zirconium/silica beads, metal beads, plastic beads, and/or sand. Thelysed material may likewise take a variety of forms, for exampleorganelles-nuclei, mitochondria, lysosomes, chloroplasts, endoplasmicreticulum, etc.

Various embodiments of the material separation and/or lysis apparatusand systems may, for example, operate in: 1) a batch mode, 2)flow-through stop or semi-batch mode, or 3) continuous flow-throughmode. In batch mode, a container having a chamber holding a sample ofmaterial to be separated or lysed is located in a holder and oscillated.The container is removed after sufficient oscillation and the separatedand/or lysed material recovered. In the flow-through stop or semi-batchmode, a sample of material to be separated or lysed flows into to fillthe chamber. The container is then oscillated until sufficientlyseparated and/or lysed. The chamber is evacuated of the separated and/orlysed material. In the flow-through mode, a sample of material to beseparated and/or lysed flows through the chamber of the container duringoscillation at a desired flow rate, providing a desired or definedresidence time within the chamber. In the flow-through stop orsemi-batch mode, the sample may abutted by an immiscible liquid or gasand the chamber may be evacuated by a blast of a fluid, for example aliquid or a gas.

At least some of the embodiments take advantage of the understandingthat the forces responsible for mechanical rupture of biological samplesscale with the oscillation frequency squared, and that by employingrelatively small sample sizes, the various embodiments described hereincan achieve relatively higher frequencies than commercially availableapparatus, resulting in rapid and efficient lysis. Various specificembodiments will now be discussed.

At least some of the embodiments take advantage of a recently identifiedproperty of material to undergo an “anti-centrifugal” force whenoscillated at a sufficiently high frequency, which frequency is afunction of various characteristics of the particles. Such may beadvantageously employed to change a direction of motion of particles orto achieve a direction of separation not previously thought to beachievable. Such may be employed with a variety of materials and is notlimited or restricted to lysing.

FIGS. 1A-1C and 2A-2C show an apparatus 10 operable to performseparation and/or lysing on a material to be separated and/or lysedcontained in a container 12, according to one illustrated embodiment. Insome embodiments, off-the-shelf vials and tubes may be employed as thecontainer 12 to hold specimens of material to be separated and/or lysedand the lysing particulate material or other material, for example PCRor Eppendorf tubes. While illustrated in FIGS. 1A-1C and 2A-2C in abatch mode, the separation and/or lysis apparatus 10 may be used in aflow-through stop or semi-batch mode or in a continuous mode asillustrated in FIG. 4.

The container 12 may be removably coupled to an arm 14 via a holder 16.The holder 16 may take a variety of forms. For example, the holder 16may take the form of a U-shaped clamp or other member. The holder 16 mayinclude a fastener (e.g., screw, bolt, etc.) 16 a operable to secure theholder 16 in a container securing configuration. Alternatively, theholder 16 may be resilient and biased into the container securingconfiguration.

The arm 14 may be coupled to pivot about an axle 18 such that thecontainer 12 oscillates along an arcuate path 20. Oscillation along anarcuate path 20 achieves confined periodic flow fields with angularaccelerations that provide strong particulate flow fields and largeshear rates between beads in a liquid solution or slurry. Experiments bythe applicants have demonstrated that miniaturized geometries canprovide superior lysis through the application of high frequencies(e.g., greater than approximately 100 Hz). Since the relative forces onnon-neutral density beads in a liquid scale according to ω²r, where ωrepresents angular velocity and r is the distance of a bead from thecenter of rotation, a small increase in angular speed can allow for asubstantial decrease in size to attain similar performance. Linearoscillatory motions, even at high frequencies result in little lysis ofbiological samples, while those with an arc motion may achieve lysisthat is superior to commercially available bead-based lysis apparatus.High-speed movies clearly show that linear motions result in periodicconcentration of beads followed by expansion of beads away from oneanother, but relatively little relative motion of beads that is notalong the axis of motion. In contrast, where a container oscillates inan arc, the beads are seen to compress to higher density just as astrong swirl is induced, resulting in very effective lysing. Collisionsand shearing provided by the relative motion of the suspended beadscontribute to the high efficiency of the lysing.

The arm 14 may be a rigid arm, i.e., an arm that does not appreciablybend during oscillation with a load having a mass at least roughlyequivalent to an expected load of a container containing a material tobe lysed and a lysing particulate material. Alternatively, the arm 14may be a flexible arm, i.e., an arm that does appreciably bend duringoscillation with a load having a mass at least roughly equivalent to anexpected load of a container containing a material to be separatedand/or lysed and optionally a lysing particulate material.

As best illustrated in FIGS. 2A-2C and 3 in which a cover plate 24 isremoved, the arm 14 may be driven via a motor 22 and a drive mechanism26, which may take the form of a four-bar linkage. In particular, ashaft 28 of the motor 22 drives a first member such as a bar, here inthe form of eccentric cam 30. The eccentric cam 30 is received in a bore32 of a second member or connecting arm 34. The connecting arm 34 isdrivingly coupled to the holder 16 by the axle 18 of a rocker arm 36.The drive mechanism 26 provides a low cost, reliable mechanism torealize relatively high frequency oscillatory motion along the arcuatepath 20. While such frequencies may not be considered high for othertypes of devices, of instance rotating devices or ultra-sonic devices,such frequencies are considered high oscillating type devices.

FIG. 4 shows a flow-through separation and/or lysis system 400 accordingto one illustrated embodiment. As described in more detail herein, theflow-through separation and/or lysis system 400 may be operated in aflow-through stop or semi-batch mode, or in a continuous flow mode.

The flow-through system 400 includes a separation and/or lysingapparatus 410 and a container 412, which may be similar to thosedescribed in previous embodiments. For example, the separation and/orlysing apparatus 410 may include an arm 414 and holder 416 to hold thecontainer 412 as the container pivotally oscillates about an axle 418.

The flow-through separation and/or lysis system 400 may include anupstream subsystem 438 to deliver material to be separated and/or lysed.For example, the upstream subsystem 438 may include a pump 440 operableto pump or otherwise deliver material to be separated and/or lysed tothe container 412. The upstream subsystem 438 may also include areservoir 442 that holds the material to separated and/or lysed.

The upstream subsystem 438 may additionally or alternatively include amechanism to collect material to be separated and/or lysed, for examplea sampling apparatus 439. The sampling apparatus 439 may be manuallyoperated or may be automatic. The sampling apparatus 439 may, forexample, sample the ambient environment, for example the air oratmosphere, water or fluids, soil or other solids. The samplingapparatus 439 may include a vacuum or mechanism to create a negativepressure to extract a sample. The sampling apparatus 439 may include anactuator, for example an arm with a shovel or broom to retrieve samples.The sampling apparatus may include an actuator, for example a needle andsyringe to example samples.

The material to be separated and/or lysed may be delivered via one ormore conduits, for example, a tube 444 a to an entrance 446 a of thecontainer 412. The tube 444 a may be reinforced at one or both ends, forexample, being reinforced with multiple layers of concentricallyarranged tubes 448 a. The tube 444 a may have a length L₁ that issufficiently long to allow the container 412 and arm 414 to oscillate,while being sufficiently short as to prevent resonance in the tube. Thelength L₁ would be a function of the density, the rigidity, or theattachment method of the tube 444 a as well as the density, mass and/orrigidity of any material to be separated and/or lysed carried therein.

The flow-through separation and/or lysis system 400 may further includea downstream analysis subsystem 449. The downstream analysis subsystem449 may include one or more downstream analysis apparatus 450. Thedownstream analysis apparatus 450 may take any of a variety of forms.For example, the downstream analysis apparatus 450 may include a nucleicacid amplification instrument, electron-microscope, western blottingapparatus, mass spectrometer, gas chromatograph, etc.

The downstream analysis subsystem 449 may further include one or morecomputing systems 452 communicatively coupled to the downstream analysisapparatus 450. The computing system 452 may be coupled to one or morenetworks 453, for example a local area network (LAN), a wide areanetwork (WAN) such as the Internet, and/or a wireless wide area network(WWAN). The computer system 452 may provide information about theresults of an analysis performed on separated and/or lysed material viathe network 453. For example, the computing system 452 may automaticallyprovide an alert or other message to suitable system based on theresults of the analysis. Such may, for example, be used to provide analert when a toxic or dangerous substance or condition is detected.

The downstream analysis apparatus 450 may be fluidly communicativelycoupled to an exit 446 b of the container 412 via one or more conduits,for example, tube 444 b. The tube 444 b may be reinforced at one or bothends, for example, by one or more concentrically arranged lengths oftube 448 b. The tube 444 b may have a length L₂ that is sufficientlylong as to allow the container 412 and arm 414 to oscillate freely whilebeing sufficiently short as to prevent resonance of the tube 444 b. Thelength L₂ may be based on the density, the rigidity, or the attachmentmethod of the tube 444 b as well as a density, mass and/or rigidity ofany material carried therein.

The flow-through separation and/or lysis system 400 may further includeone or more control systems 454. The control system 454 may take theform of one or more motor controllers and/or computing systems. Thecontrol system 454 may be configured to operate the flow-through system400 in a flow-through stop or semi-batch mode and/or in a flow-throughcontinuous flow mode. The control systems 454 may, for example, becommunicatively coupled to control the separation and/or lysingapparatus 410 and/or pump 440.

The flow-through system 400 provides a number of advantages over batchbased apparatus. For example, some types of beads may have an affinityfor certain bio-products that are released on lysis, so some of the cellcontents may be “lost” due to adsorption on the bead surfaces. Theflow-through design may advantageously automatically elute the adsorbedbiomolecules. It also avoids difficult or additional acts that may berequired in batch mode configurations to evacuate the chamber. Forexample, the flow-through embodiments may eliminate any possible need toblast the chamber with a fluid such as air to clear the chamber of theseparated and/or lysed material.

FIG. 5 shows a container 512 according to one illustrated embodiment.

The container 512 may have an entrance 546 a to provide fluidcommunication from an exterior 560 of the container to a chamber 562 ofthe container 512. The container 512 may include an exit 546 b providingfluid communication between the exterior 560 and the chamber 562 of thecontainer 512. A first tube 544 a may be coupled to the container 512 toprovide material to be lysed 564 to the chamber 562 via the entrance 546a. As noted previously, the tube 544 a may be reinforced, for example,with one or more layers of concentrically arranged tubing 548 a. Asecond tube 544 b may be coupled to the container 512 via the exit 546 bto remove lysed material 566 via the exit 546 b. In some embodiments,the container 512 may include attachment structures to attach orotherwise couple or secure the tubes 544 a, 544 b. For example, thecontainer 512 may include a ribbed nipple 568 a at the entrance 546 aand/or a ribbed nipple 568 b at or proximate the exit 546 b.

The container includes lysing material 570. The lysing material 570 maytake a variety of forms, for example, a plurality of beads. The beadsmay take a variety of forms including one or more of ceramic beads,glass beads, zirconium beads, zirconium/silica beads, metal beads,plastic beads, and/or sand. The beads may have a variety of diameters,for example, between approximately 10 microns and approximately 600microns.

In the flow through embodiments, the container 512 may include a firstfilter 572 a positioned relatively proximate the entrance 546 a and asecond filter 572 b positioned relatively proximate the exit 546 b. Thefirst and second filters 572 a, 572 b form a particulate retainment area574 in which the lysing particulate material 570 is retained. Inparticular, the filters 572 a, 572 b may have a plurality of openingssized to substantially pass the material to be lysed 564 and the lysedmaterial 566, respectively, while blocking the particulate lysingmaterial 570. The container 512 may include one or more structures, forexample, tabs or annular ridges 576 a, 576 b to retain the first andsecond filters 572 a, 572 b in place. Filters may, for example take theform of nylon or stainless steel mesh filter.

The embodiments of FIGS. 1A-5 may advantageously allow extremely highpacking densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. Additionally oralternatively, these embodiments may advantageously have essentially noair in the chamber. As used herein, essentially no air means that thechamber is free of air other than small bubbles which may beunintentionally entrapped in the chamber. Such may increase lysingefficiency and prevent undesirable heating of the system from frictionassociated with liquid-air contact line motions.

FIG. 6 shows a method 600 of operating an apparatus such as thatillustrated in FIGS. 1A-4 to lyse material, according to one illustratedembodiment.

At 602, material to be lysed is received in the chamber of thecontainer. The chamber may already hold lysing particulate material. At604, the container is oscillated along an arcuate path. The oscillationproduces large variations in movement between respective ones of thelysing particulate material. Such variations are more pronounced than intranslational or rotational movements. At 606, the lysed material isremoved from the chamber of the container.

FIG. 7 shows a method 700 of pumping material to be lysed in aflow-through lysing system such as the one of FIG. 4, according to oneillustrated embodiment.

At 702, the material to be lysed is pumped into the chamber of thecontainer.

FIG. 8 shows a method 800 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to oneillustrated embodiment.

At 802, the material to be lysed is intermittently pumped into thechamber of the container while the container is oscillated. Such issuitable for the flow-through stop or semi-batch mode.

FIG. 9 shows a method 900 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 902, the material to be lysed is intermittently pumped into thechamber such that the material to be lysed spends a sufficient time inthe chamber to achieve a desired level of lysing. Thus, if is determinedthat 30 seconds of oscillation achieves a desired level of lysing, thepump may be intermittently operated to load the chamber with material tobe lysed approximately every 30 seconds. Oscillation times of fewseconds or tenths of seconds may be suitable. Such operation is suitablefor the flow-through stop or semi-batch mode.

FIG. 10 shows a method 1000 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 1002, the material to be lysed is intermittently pumped into thechamber such that the chamber is completely evacuated of the lysedmaterial during each cycle of the intermittent pumping. Such is suitablefor the flow-through stop or semi-batch mode.

FIG. 11 shows a method 1100 of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 1102, the chamber is evacuated of the lysed material during eachcycle of the intermittent pumping by pumping into the chamber morematerial to be lysed. Such is suitable for the flow-through stop orsemi-batch mode.

FIG. 12 shows a method 1200 of operating a lysing apparatus such as thatof FIG. 4, according to another illustrated embodiment.

At 1202, the chamber is evacuated of the lysed material each cycle ofthe intermittent pumping by pumping an inert fluid into the chamber. Theinert fluid may take the form of a liquid or gas, and may be immisciblewith the lysed material or material to be lysed. Such is suitable forthe flow-through stop or semi-batch mode.

FIG. 13 shows a method 1300 of operating a continuous lysing apparatus,according to one illustrated embodiment.

At 1302, the material to be lysed is continuously pumped into thechamber of the container while the container is oscillated. Such issuitable for the flow-through continuous mode.

FIG. 14 shows a method 1400 of operating a flow-through lysingapparatus, according to another illustrated embodiment.

At 1402, a flow rate of the pumping of the material to be lysed isadjusted based at least in part on the length and free volume of thechamber such that the material to be lysed spends sufficient time in thechamber (i.e., desired or defined residence time) to achieve a desiredlevel of lysing. Such is suitable for the flow-through continuous mode.

FIG. 15 shows a method 1500 of operating a flow-through lysingapparatus, such as that of FIG. 4, according to another illustratedembodiment.

At 1502, the lysed material removed from the chamber of the container isdirected to at least one analysis device. At 1504, the lysed material isanalyzed. Analysis may take a variety of forms, for example analysiswith electron-microscope, western blotting, mass spectrometry, gaschromatography, etc. Such is suitable for any of the modes, andparticularly suited to the flow-through modes.

FIG. 16 shows a flow-through lysing apparatus 1600 according to anotherillustrated embodiment. As described in more detail herein, the flowthrough lysis system 1600 may be operated in a flow-through stop orsemi-batch mode, or in a continuous flow mode.

The flow-through lysing apparatus 1600 includes a container 1602 havinga chamber 1604, and a micromotor 1606 coupled to drive an impeller 1608.

As illustrated, the chamber 1604 may have a first opening 1604 a thatserves as an entrance providing fluid communication from an exterior1610 of the container 1602 to the chamber 1604. Also as illustrated, thechamber 1604 may have a second opening 1604 b that serves as an exit,providing fluid communication from the chamber 1604 to the exterior1610. The container 1602 may further have a third opening 1604 c sizedto receive the impeller 1608 and to sealingly engage an outer portion ofthe micromotor 1606. Some embodiments may include a bushing or O-ring toform or enhance the sealing between the micromotor 1606 and thirdopening 1604 c.

A first coupler 1610 a may include a stem 1612 a sized to be sealinglyreceived in the opening 1604 a to provide fluid communication into thechamber 1604. The stem 1612 a may be threaded with the hole 1604 ahaving a complementary thread. The first coupler 1610 a may include anattachment structure, for example, a ribbed nipple 1614 a to secure atube 1616 a and provide a flow of material to be lysed to the chamber1604. An O-ring 1618 a, or other similar structure, may enhance a sealbetween a flange of the first coupler 1610 a and the container 1602.

A second coupler 1610 b may include a stem 1612 b sized to be sealinglyreceived in the opening 1604 b to provide fluid communication into thechamber 1604. The stem 1612 b may be threaded with the hole 1604 bhaving a complementary thread. The second coupler 1610 b may include anattachment structure, for example, a ribbed nipple 1614 b to secure atube 1616 b and provide a flow of material to be lysed to the chamber1604. An O-ring 1618 b, or other similar structure, may enhance a sealbetween a flange of the second coupler 1610 b and the container 1602.

Filters 1619 a, 1619 b may be positioned in the chamber to retain lysingparticulate material therebetween. The filters 1619 a, 1619 b may, forexample, take the form of nylon mesh filters with 50 micron openingsmounted to suitable fittings.

The micromotor 1606 may, for example, take the form of a micromotorhaving a 4 mm diameter, and may be capable of driving the impeller athigh speed, for example approximately 50,000 RPM, when not in thepresence of liquid and beads. The impeller 1608 may be a nylon oracrylic impeller having a number of vanes. The vanes may be straight,without curvature or angle of attachment, such that movement of materialis primarily circumferential. Should axial/horizontal movement of thematerial through the chamber be desirable, for example in a flow-throughmode (e.g., FIGS. 16 and 17), such axial or flow movement comes frompumping and not from rotation of the impeller. This allows more precisecontrol over amount of time that the material remains in the chamber andhence is subject to lysis. The vanes may, for example, produce aperiodic flow at a frequency nearly 5 times as high as the embodimentsof FIGS. 1A-4, however with a smaller amplitude of motion.

The lysing apparatus 1600 may also include a controller 1620 coupled tocontrol the micromotor 1606. The controller 1620 may, for exampleinclude a motor controller and/or a programmed general purpose computingsystem, a special purpose computer, an application specific integratedcircuit (ASIC) and/or field programmable gate array (FPGA). Thecontroller 1620 may for example, be programmed or configured to causethe motor to pulsate. Pulsating may increase the effectiveness of thelysing.

FIG. 17 shows a flow-through lysing system 1700 according to oneillustrated embodiment. As described in more detail herein, theflow-through lysis system 1700 may be operated in a flow-through stop orsemi-batch mode, or in a continuous flow mode.

The flow-through lysing system 1700 includes a container 1702 having achamber (not illustrated in FIG. 17), openings 1704 a, 1704 c (only twoillustrated), and a micromotor 1706 coupled to an impeller (not shown inFIG. 17). The opening or entrance 1704 may be fluidly communicativelycoupled to a pump 1720 that delivers material to be lysed from areservoir 1722 via a first conduit or tube 1716 a. A second opening orexit may deliver lysed material to one or more downstream analysisapparatus 1724 via one or more conduits such as tubes 1716 b. Aspreviously noted, downstream analysis may take a variety of forms, forinstance nucleic acid amplification, electrophoresis, western blotting,mass spectrometry, gas chromatography, etc. The downstream analysisapparatus 1724 may be communicatively coupled to one or more computingsystems 1726. The flow-through lysing system 1700 may also include oneor more control systems 1728 which may control the micromotor 1706and/or pump 1720. The control system 1728 may for example synchronizethe pumping and oscillation, for example to implement a flow-throughstop or semi-batch mode. The control system 1728 may for example controlthe pumping to attain a desired or defined residence time of thematerial in the chamber to achieve a desired or defined level of lysing,for example to implement a flow-through continuous mode.

The embodiments of FIGS. 16 and 17 may advantageously allow extremelyhigh packing densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. Additionally oralternatively, these embodiments may advantageously have essentially noair in the chamber. As used herein, essentially no air means that thechamber is free of air other than small bubbles which may beunintentionally entrapped in the chamber. Such may increase lysingefficiency and prevent undesirable heating of the system from frictionassociated with liquid-air contact line motions.

FIG. 18 shows a lysing system 1800 according to another illustratedembodiment. The lysing system 1800 is particularly suitable for batchmode lysing operations.

The lysing system 1800 includes a container 1802 having a chamber 1804that has a single opening 1804 a to provide fluid communication with anexterior of the container 1802. The apparatus 1800 includes a micromotor1806 coupled to drive an impeller 1808 that is received in the chamber1804. A portion of the micromotor 1806 is sized to form a sealingengagement with the container 1802 to seal the opening 1804 a. Someembodiments may include one or more bushings or O-rings (not shown) toensure the seal.

Initially, the chamber 1804 is packed with material to be lysed 1810 andlysing particulate material 1812. After rotation of the impeller 1808,for a sufficient length of time, the chamber 1804 contains material thathas been lysed and the lysing particulate material 1812. The micromotor1806 and impeller 1808 may then be removed and the lysed material may beextracted, for example using a pipette 1814. The chamber 1804 of thebatch mode embodiments may not be as densely packed as in flow-throughembodiments since room may be required for the apparatus to withdraw thelysed material.

In some embodiments, off-the-shelf vials and tubes may be employed asthe container 1802 to hold specimens of material to be lysed and thelysing particulate material, for example PCR or Eppendorf tubes.

The embodiment of FIG. 18 may advantageously allow extremely highpacking densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. This embodimentis less likely to ensure that there is essentially no air in the chambersince room may be required for receiving the withdrawal apparatus (e.g.,pipette). However, where possible, elimination of air in the chamber mayincrease lysing efficiency and prevent undesirable heating of the systemfrom friction associated with liquid-air contact line motions.

FIG. 19 shows a method 1900 of operating a flow-through lysing apparatusand/or system according to one illustrated embodiment. Such may beuseful in a flow-through stop or semi-batch mode or in a flow-throughcontinuous mode.

At 1902, material to be lysed is received in the chamber of a containervia an entrance. The chamber may already hold lysing particulatematerial. At 1904, the micromotor drives the impeller to cause thelysing particulate material to lyse the material to be lysed. At 1906,material that has been lysed is expelled from the chamber of thecontainer via an exit.

FIG. 20 shows a method 2000 of evacuating material that has been lysedfrom a chamber, according to one illustrated embodiment.

At 2002, the material that has been lysed may be expelled via a firstfilter position before the exit in a flow path of material through theapparatus or system.

FIG. 21 shows a method 2100 of receiving material to be lysed in achamber, according to another illustrated embodiment.

At 2102, the material to be lysed is received in the chamber via asecond filter positioned following the entrance of the chamber in theflow path through the apparatus or system.

FIG. 22 shows a method 2200 of pumping material to be lysed into achamber, according to another illustrated embodiment.

At 2202, the material to be lysed is intermittently pumped into thechamber via the entrance. Such may be particularly suitable forflow-through stop or semi-batch mode operation.

FIG. 23 shows a method 2300 of pumping material to be lysed into achamber, according to one illustrated embodiment.

At 2302, the material to be lysed is continuously pumped into thechamber of the container via the entrance, at a flow rate that providesfor a resident time of the material to be lysed in the chamber that issufficiently long to achieve a desired or defined level of lysing. Themicromotor may continuously drive the impeller to lyse the material.Such may be particularly suitable for flow-through continuous modeoperation.

FIG. 24 shows a method 2400 of operating an impeller of a lysing system,according to one illustrated embodiment.

At 2402, the micromotor pulsatingly drives the impeller. Pulsations maybe achieved by varying a voltage or current delivered to the micromotor.Pulsating may achieve a higher efficiency of lysing, thereby increasingthroughput or decreasing time required to achieve a desired or definedlevel of lysing.

FIG. 25 shows a method 2500 of operating an impeller of a lysing systemaccording to one illustrated embodiment.

At 2502, the micromotor drives the impeller at greater than 10,000 RPMin the presence of liquid and beads. Driving the impeller at arelatively high speed achieves a desired or defined level of lysing.

FIG. 26 shows a method 2600 of replacing a micromotor of a lysing systemaccording to one illustrated embodiment.

At 2602, the micromotor may be replaced with a new micromotor. At 2604,the old micromotor may be disposed or recycled. This may be particularlyuseful since it is difficult to seal the internal elements (e.g., rotor,stator) of the high speed micromotor from exposure to the ambientenvironment, thus the micromotors may fail more frequently than in otherembodiments or environments.

FIG. 27 shows a method 2700 of operating a batch based lysing apparatusaccording to one illustrated embodiment. The method 2700 may beparticularly useful for use with the embodiment of FIG. 18.

At 2702, material to be lysed is received in a chamber of a firstcontainer via an entrance. The chamber may already hold a lysingparticulate material or the lysing material may be provided into thechamber with or after the material to be lysed.

At 2704, an impeller is located in the chamber of the first container.At 2706, the entrance to the first container is closed or sealed with amicromotor. At 2708, the micromotor drives the impeller to circulate thematerial to be lysed and the lysing particulate material. The micromotormay drive the impeller for a sufficient length of time at a sufficientspeed until a desired or defined level of lysing has occurred.

FIG. 28 shows a method 2800 of operating a lysing apparatus according toone illustrated embodiment. The method 2800 may be particularly usefulfor use with the embodiment of FIG. 18.

At 2802, the micromotor may be removed from the entrance of the firstcontainer. At 2804, the material that has been lysed is removed from thechamber of the first container via the entrance.

FIG. 29 shows a method 2900 of removing material that has been lysedaccording to one illustrated embodiment.

At 2902, the material that has been lysed may be withdrawn using apipette.

FIG. 30 shows a method 3000 of operating a lysing apparatus according toanother illustrated embodiment.

At 3002, the micromotor may be reused with one or more additionalcontainers. It is noted that the micromotor, particularly when operatedat high speed, may not be particularly well protected from the materialto be lysed, lysing particulate material, or lysed material.Consequently, the micromotor may wear out. In many applications themicromotor may be employed to lyse multiple samples before failing.

FIG. 31 shows data on efficiency of lysis using an apparatus similar tothat of FIG. 4.

A first curve 3102 represents measured fluorescence versus time ofoscillation using an embodiment similar to that illustrated in FIG. 4.Fluorescence is proportional to the amount of nucleic acid released fromcells. A second curve 3105 represents measured fluorescence versus timeof oscillation using a commercially available “MINI-BEADBEATER-1 productfrom Biospec Products, Inc. of Bartlesville, Okla. As seen by comparisonof the first curve 3102 and second curve 3105, the embodiment of FIG. 4causes the release of cell contents more efficiently than thecommercially available apparatus.

FIG. 32 illustrates a dependency of lysis efficiency on the frequency.

A curve 3202 appears to indicate a nearly quadratic dependence of thedegree of lysis on frequency as controlled by changes to the appliedvoltage for a fixed amount of time.

FIG. 33 shows data representing spore lysis as a function of time for anembodiment similar to that illustrated in FIGS. 16 and 17.

The curves 3302, 3304 illustrate that the time to saturation iscomparable to that of the embodiments of FIG. 4, but with peakefficiency of only 80%. The power required for this efficiency was only400 mW, which is lower than the power used for various otherembodiments.

FIG. 34 shows a material separation apparatus 3410 according to oneillustrated embodiment.

The material separation apparatus 3410 has a base 3412. The materialseparation apparatus 3410 includes an actuator in the form of anelectric motor 3414 and a transmission or drive mechanism 3416 coupledto the base 3412. The electric motor 3414 is selectively operable todrive the drive mechanism 3416 to oscillatingly angularly rotate (i.e.,oscillating pivot) a container 3418, about an axis of rotation 3420 asindicated by double headed arrow 3422. Notable in this embodiment, theaxis of rotation 3420 passes through a portion of the container 3418.The container 3418 has an interior 3424 that holds material 3426. Thematerial 3426, is material to be separated at a first time, and isseparated material at a second time.

The drive mechanism 3416 may include a first drive member 3430 that isrotated by a drive shaft 3432 of the motor 3414. A second drive member3434 may be coupled to the first drive member 3430 may a connecting rodor member 3436 such that the second drive member eccentrically rotatesthe container 3418. Other drive members may be employed, for exampleeccentric gears or cams. The second drive member 3434 is coupled to aholder 3436 to which the container 3418 is removably attached orpermanently fixed.

FIGS. 35A and 35B show a container 3500 according to one illustratedembodiment.

As illustrated, the container 3500 may have an oval or circular outerperiphery. The container 3500 may be mounted concentrically with respectto an axis of rotation 3502, for oscillating angular rotation thereaboutas indicated by double headed arrow 3504. Thus, the axis of rotation3502 passes through a portion of the container 3500.

The container 3500 may include at least one port 3506 to transfermaterial between an interior 3508 of the container 3500 and an exterior3510 thereof. The container 3500 may include one or more filters (nowshown), which may, for example take the form of nylon or stainless steelmesh filter. One or more of the ports, collectively 3506, may include avalve and/or filter.

FIGS. 36A and 36B show a container 3600 according to one illustratedembodiment.

As illustrated, the container 3600 may have a rectangular or squareouter periphery. The container 3600 may be mounted concentrically withrespect to an axis of rotation 3602, for oscillating angular rotationthereabout as indicated by double headed arrow 3604. Thus, the axis ofrotation 3602 passes through a portion of the container 3600.

The container 3600 may include at least one port 3606 to transfermaterial between an interior 3608 of the container 3600 and an exterior3610 thereof. The container 3600 may include one or more filters (nowshown), which may, for example take the form of nylon or stainless steelmesh filter. One or more of the ports, collectively 3606, may include avalve and/or filter.

FIGS. 37A and 37B show a container 3700 according to one illustratedembodiment.

As illustrated, the container 3700 may have an annular cross-sectionwith an oval or circular outer periphery 3700 a and an oval or circularinner periphery 3700 b. The container 3700 may be mounted concentricallywith respect to an axis of rotation 3702, for oscillating angularrotation thereabout as indicated by double headed arrow 3704. Thus, theaxis of rotation 3702 passes through a portion of the container 3700.

The container 3700 may include a number of outer ports 3706 a, 3706 b totransfer material between an interior 3708 of the container 3700 and anexterior 3710 thereof. In particular, the outer ports 3706 a, 3706 b maybe formed in the outer periphery 3700 a of the container 3700. Thecontainer 3700 may include a number of inner ports 3706 c, 3706 d totransfer material between the interior 3708 of the container 3700 andthe exterior 3710 thereof. In particular, the inner ports 3706 c, 3706 dmay be formed in the inner periphery 3700 b of the container 3700. Thecontainer 3700 may include one or more filters (now shown), which may,for example take the form of nylon or stainless steel mesh filter. Oneor more of the ports, collectively 3706, may include a valve and/orfilter.

FIGS. 38A and 38B show a container 3800 according to one illustratedembodiment.

As illustrated, the container 3800 may have an annular cross-sectionwith an oval or circular outer periphery 3800 a and an oval or circularinner periphery 3800 b. The container 3800 may be mounted concentricallywith respect to an axis of rotation 3802, for oscillating angularrotation thereabout as indicated by double headed arrow 3804. Thus, theaxis of rotation 3802 passes through a portion of the container 3800.

The container 3800 may include a number of outer ports 3806 a, 3806 b totransfer material between an interior 3808 of the container 3800 and anexterior 3810 thereof. In particular, the outer ports 3806 a, 3806 b maybe formed in the outer periphery 3800 a of the container 3800. Thecontainer 3800 may include a number of inner ports 3806 c, 3806 d totransfer material between the interior 3808 of the container 3800 andthe exterior 3810 thereof. In particular, the inner ports 3806 c, 3806 dmay be formed in the inner periphery 3800 b of the container 3800. Thecontainer 3800 may include one or more filters (now shown), which may,for example take the form of nylon or stainless steel mesh filter. Oneor more of the ports, collectively 3806, may include a valve and/orfilter.

FIGS. 39A and 39B show a container 3900 according to one illustratedembodiment.

As illustrated, the container 3900 may have an oval or circular crosssection with an oval or circular outer periphery 3900 a and an oval orcircular inner periphery 3900 b. The container 3900 may be mounted foroscillating angular rotation about an axis of rotation 3902 as indicatedby double headed arrow 3904. Thus, the axis of rotation 3902 does notpass through any portion of the container 3900.

The container 3900 may include a number of outer ports 3906 a totransfer material between an interior 3908 of the container 3900 and anexterior 3910 thereof. The container 3900 may include a number of innerports 3906 b to transfer material between the interior 3908 of thecontainer 3900 and the exterior 3910 thereof. In particular, the outerport 3906 a may spaced relatively farther from the axis of rotation 3902than the inner port 3906 b. The container 3900 may include one or morefilters (now shown), which may, for example take the form of nylon orstainless steel mesh filter. One or more of the ports, collectively3906, may include a valve and/or filter.

FIGS. 40A and 40B show a container 4000 according to one illustratedembodiment.

As illustrated, the container 4000 may have an oval or circular crosssection with an oval or circular outer periphery 4000 a and an oval orcircular inner periphery 4000 b. The container 4000 may be mounted foroscillating angular rotation about an axis of rotation 4002 as indicatedby double headed arrow 4004. Thus, the axis of rotation 4002 dos notpass through any portion of the container 4000.

The container 4000 may include a number of outer ports 4006 a totransfer material between an interior 4008 of the container 4000 and anexterior 4010 thereof. The container 4000 may include a number of innerports 4006 b to transfer material between the interior 4008 of thecontainer 4000 and the exterior 4010 thereof. In particular, the outerport 4006 a may spaced relatively farther from the axis of rotation 4002than the inner port 4006 b. The container 4000 may include one or morefilters (now shown), which may, for example take the form of nylon orstainless steel mesh filter. One or more of the ports, collectively4006, may include a valve and/or filter.

FIG. 41 shows a method 4100 of operating an apparatus to separatematerials, according to one illustrated embodiment.

At 4102, a material to be separated is received in a container. Thematerial may, for example, include a particulate material in asuspension.

At 4105, the container is oscillating angularly rotated at a highfrequency. Such may be implemented by supplying power to a motor todrive a drive mechanism coupled to the container.

At 4106, at least some of the separated material is removed from thecontainer. For example, the relatively dense or heavier material may beremoved. The relatively dense or heavier material may collect at aportion of the interior of the container that is relatively closer to anaxis of rotation than other portions of the interior of the container.Thus, such dense or heavier material may be removed, for instance, viaan inner port of the container. Also for example, the relatively lessdense or lighter material may be removed. The relatively less dense orlighter material may collect at a portion of the interior of thecontainer that is relatively farther from an axis of rotation than otherportions of the interior of the container. Thus, such less dense orlighter material may be removed, for instance, via an outer port of thecontainer. The separated material being removed may pass through one ormore filters to further separate materials.

FIG. 42 shows a method 4200 of operating an apparatus to separatematerials, according to one illustrated embodiment.

At 4202, the material to be separated is pumped into the container.

FIG. 43 shows a method 4300 of operating an apparatus to separatematerials, according to one illustrated embodiment.

At 4302, the material to be separated is intermittently pumped into thecontainer while oscillating the container.

FIG. 44 shows a method 4400 of operating an apparatus to separatematerials, according to one illustrated embodiment.

At 4402, at least some of the separated material removed from thecontainer is directed to at least one analysis device. Such may beaccomplished using gravity flow, pumps, valves, etc.

FIG. 45 shows a method 4500 of operating an apparatus to separatematerials, according to one illustrated embodiment.

At 4502, the container is evacuated of the separated materials using aninert fluid. For example the container may be flushed with an inert gasor liquid. Such may prepare the container for a next specimen, sample orbatch of material to be separated.

To summarize, apparatus and methods cause separation of particles (e.g.,cells, bio-molecules, etc.) in a fluid suspension by imparting angularoscillations to the fluid container, which essentially undergoesoscillatory rigid-body rotation. Particles whose density is differentfrom the fluid can be separated radially similar to centrifugation.However, the direction of particle motion and accumulation canunexpectedly be opposite to ordinary centrifugation. One can thuscollect the relatively heavy or denser particles near the rotation axiswhile the relatively light or less dense particles are thrown away fromthe axis of rotation. In contrast, in ordinary centrifugation, particlesdenser than the fluid move away from the rotation axis.

As taught here, it is shown that if instead of rotating steadily such asin an ordinary centrifuge, the container undergoes high-frequency,purely oscillatory, angular rotation, dense or relatively heavyparticles can be made to move toward the rotation axis while light orrelatively less dense particles can be moved away from the axis ofrotation.

Thus, such provides an approach to separating particles based on theirdensity difference (but also dependent upon their size) in a mannersimilar to a centrifuge. However, the direction of particle migrationcan be manipulated (for instance by changing the frequency ofoscillations) to be opposite to what one expects in an ordinarycentrifuge. Such can potentially be applied to separation of red andwhite blood cells or other bio-particles or bio-molecules. In additionto particle separation and concentration, one can envision using suchfor re-suspension of particles that have already been separated in anordinary centrifuge. For instance, heavy particles are centrifuged out,but are then re-suspended by putting the container in an oscillatoryangular rotation mode, rather than in its original steady rotation.

In practice, a particle suspension is introduced into and completelyfills a container (for instance a chamber having a square cross-section,thin side walls, and a top cover) and the container is made to undergooscillatory angular rotations about an axis perpendicular to thecenterline of the container (e.g. center of the square cross-section).The frequency and amplitude of oscillations can be varied. Particlesmigrate radially and collect near the rotation axis or near the sidewalls, depending on their density and size.

The above approach is based on a theoretical analysis of particlemotion, set out below. The theoretical analysis neglects some effectsthat are assumed to be of minor importance (e.g. Basset history-integralforces and lift forces on the particles as well as hydrodynamicinteractions among the particles and between the particles and thewalls). These effects may end up being significant and may modify thecurrent predictions. Experimental verification is planned.

Applicants have observed that linear sliding motion is not as effectiveat lysing spores as the “wagging” or oscillatory motion described hereinand in U.S. provisional patent application Ser. No. 61/020,072 filedJan. 9, 2008, which is incorporated by reference herein in its entirety.

The equations of motion for a bead include:

$\begin{matrix}{{m_{p}\frac{V}{t}} = {{m_{f}\frac{Du}{Dt}} - {\frac{1}{2}{m_{f}\left( {\frac{V}{t} - \frac{Du}{Dt}} \right)}} - {6{\pi\mu}\; {a\left( {V - u} \right)}} + {\left( {m_{p} - m_{f}} \right)g}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where the first term after the equal sign represents pressure stress,the second term represents added mass, the third term viscous drag andthe forth term represents gravity, but can be ignored or neglected.

Where cartridge displacement is represented by:

Δ sin(ωt)i  Equation 2

And fluid acceleration is represented by:

Du/Dt=−ω ²Δ sin(ωt)i  Equation 3

The equation of motion for the bead becomes:

(m _(P)+½m _(f)){umlaut over (x)}=−3/2m _(f)ω²Δ sin (ωt)−6πμα[{dot over(x)}−ωΔ cos(ωt)]  Equation 4

with initial conditions:

x(0)=0{dot over (x)}(0)=ωΔ,  Equation 5

In moving frame and dimensionless, the equation is represented as:

{umlaut over (X)}=(1=α)sin(t)−β{dot over (X)}  Equation 6

where

$\begin{matrix}{\alpha = {{\frac{3m_{f}}{{2m_{p}} + m_{f}}\mspace{14mu} \beta} = {\frac{6{\pi\mu}\; a}{\omega \left( {m_{p\mspace{14mu} -}m_{f}\text{/}2} \right)} = {St}^{- 1}}}} & {{Equation}\mspace{14mu} 8}\end{matrix}$

and with initial conditions:

X(0)=0{dot over (X)}(0)=0  Equation 9

The solution is given by:

$\begin{matrix}{{X(t)} = {\left( {1 - \alpha} \right)\left\{ {\frac{1}{\beta} - \frac{^{{- \beta}\; t}}{\beta \left( {1 + \beta^{2}} \right)} - {\frac{1}{1 + \beta^{2}}\left\lbrack {{\sin (t)} + {\beta \; {\cos (t)}}} \right\rbrack}} \right\}}} & {{Equation}\mspace{14mu} 10}\end{matrix}$

FIG. 46 shows bead trajectory, linear oscillations.

FIG. 47 shows constant distance b/w neighboring beads.

Oscillatory rotational motion is represented by:

φ(t)=Δ sin(ωt)

Ω(t)={dot over (φ)}=ωΔ cos(ωt)

{dot over (Ω)}(t)=−ω²Δ sin(ωt)  Equations 11

And fluid acceleration by:

$\begin{matrix}{\frac{Du}{Dt} = {{\overset{.}{\Omega}r{\hat{e}}_{\theta}} - {\Omega^{2}r{\hat{e}}_{r}}}} & {{Equations}\mspace{14mu} 12}\end{matrix}$

The equations of motion are represented as:

{umlaut over (r)}=r({dot over (θ)})² =−αrΩ ²−ωβ{dot over (r)}

r{umlaut over (θ)}−2{dot over (r)}{dot over (θ)}=α{dot over(Ω)}r−ωβr({dot over (θ)}−Ω)

r(0)=r _(o)θ(0)=0{dot over (r)}(0)=0{dot over (θ)}(0)=ωΔ  Equations 13

In rotating frame and dimensionless, the equations of motion are become:

{umlaut over (r)}=−β{dot over (r)}+Δ ² r[(1−α)cos²(t)+2 cos(t)+2cos(t){dot over (δ)}+{dot over (δ)}²]

{umlaut over (δ)}=(1=α)sin(t)−β{dot over (δ)}−2({dot over (r)}/r)[{dotover (δ)}+cos(t)]

r(0)=1δ(0)=0{dot over (r)}=0{dot over (δ)}(0)=0  Equations 14

with parameters:

$\begin{matrix}{{\left( {1 - \alpha} \right) = \frac{m_{p} - m_{f}}{m_{p} + {m_{f}\text{/}2}}}{\beta = {\frac{6\pi \overset{\_}{\omega}a}{\omega \left( {m_{p} + {m_{f}\text{/}2}} \right)} = {St}^{- 1}}}} & {{Equations}\mspace{14mu} 15}\end{matrix}$

FIG. 48 represents how particles that are denser or heavier that thefluid may move toward the rotational axis rather than moving away aswould have been expected.

FIG. 49 represents the effect of a larger Stokes number, hence smallerdrag.

FIG. 50 represents convergence of neighboring beads.

An approximate may be made via a method of averaging. Where

β<√{square root over (α)}

particles move radially inward, while where

β<√{square root over (α)}

particles move radially outward.

FIGS. 51A and 51B show a lysing apparatus 5100, according to anotherillustrated embodiment.

The lysing apparatus 5100 includes a body 5102 that forms a chamber5104. The body 5102 may have an opening 5106 sized and dimensioned toreceive an impeller 5108 therethrough such that the impeller resides inthe chamber 5104. The opening 5106 may optionally receive part or all ofa drive motor, for instance a micro electric motor 5110. The electricmotor 5110 is coupled to drive the impeller 5108. The electric motor5110 is selectively operable in response to power supplied thereto. Theelectric motor 5110 may be secured in the opening 5106 via a press typefitting or interference fit. In particular, an inner wall forming theopening 5106 and/or chamber 5104 may be slightly tapered to sealingengage a side wall of the electric motor 5110 as the electric motor isadvanced through the opening 5106 and into the chamber 5104.Alternatively, or additionally, a side wall of the electric motor 5110may be slightly tapered to sealing engage a side wall of the opening5106 and/or the chamber 5104 as the electric motor 5110 is advancedthrough the opening 5106 and into the chamber 5104. Alternatively, theelectric motor 5110 and the opening 5106 and/or chamber 5104 may includecoupler structures. For instance, the electric motor 5110 and theopening 5106 and/or chamber 5104 may include threads (not shown) whichsealing mate together as the electric motor 5110 is advanced through theopening 5106 and into the chamber 5104. Alternatively, a bayonet (notshown) or lug type (not shown) coupler structure may be employed. Othersealing structures may be employed. For example, one or more gaskets,washers or O-rings (not shown) may be employed, with or without a seator peripheral ring to seat the gasket, washers or O-rings. The seal maybe a fluid tight seal and/or a gas tight seal.

The lysing apparatus includes a first port 5112 a and a second port 5112b (collectively 5112). The first and second ports 5112 include passages5114 a, 5114 b, respectively, (collectively 5114) to provide fluidcommunication with the chamber from an exterior thereof. The ports 5112may be used to as input ports to supply material to the chamber 5104and/or as output ports to remove material from the chamber 5104.

Each port 5112 may have a coupler 5116 a, 5116 b (collectively 5116)that allows selective coupling to the respective port 5112 a, 5112 b.For example, each of the ports 5112 may include a respective Luer-Lock®fitting or Luer-Slip® fitting, male or female. The Luer-Lock® orLuer-Taper® fittings allow the coupling of syringes 5118 a, 5118 b (FIG.51A, collectively 5118) to the lysing apparatus 5100. For example, afirst syringe 5118 a may be coupled to the first port 5112 a to allowsample or specimen injection, while a second syringe 5118 b may becoupled to the second port 5112 b to allow removal of a sample orspecimen after lysing (i.e., lysed material). Such may allow the passageof a sample or specimen back and forth through the chamber 5104, forinstance to enhance performance of the lysing or of DNA capture. Use ofsyringes 5118 may occur at either port 5112 a, 5112 b or at both ports5112. The advantages of using a syringe 5118 as a sample or specimendelivery system include the fact that syringes 5118 are inexpensive,disposable, and employ positive displacement of fluid for a high degreeof reliability in rapidly dispensing volumes. The Luer-Lock® designexemplifies a universal attachment that seals reliably and mates withmany devices that also have complimentary Luer-Lock® fittings.

As illustrated in FIG. 52, selectively fastenable fittings, such as theLuer-Lock® fittings, may allow multiple lysing apparatus 5100 a-5100 b(collectively, 5100, only three illustrated) to be connected insuccession. Such may advantageously be used to sequentially process asample or specimen through multiple stages. Additionally, oralternatively, lysing particulate (e.g., beads) in the differentsequential lysing apparatus 5100 may each have a respective receptivityfor different molecules. For instance, the particulate in successiveones of the sequential lysing apparatus may be conferred with receptors(e.g., binding sites) to capture different respective molecules from thesame sample or specimen. Each lysing apparatus 5100 with a differentcaptured molecule, may then be easily separated from one another, andprocessed individually using different types of elution acts or steps.

FIGS. 53A and 53B show a lysing manifold or array 5300, according to oneillustrated embodiment. The lysing manifold or array 5300 includes ablock or frame 5302 that has a plurality of positions 5304 a, 5304 h(collectively 5304, only two called out in FIG. 53A) to hold respectiveones of one or more individual lysing apparatus 5306 a-5306 h(collectively 5306, six illustrated). The individual lysing apparatus5306 may, for example, take the form of distinct lysing apparatus whichemploy a chamber that receives an impeller and electric motor, forinstance, the individual lysing apparatus 53006 may be identical orsimilar to the lysing apparatus 5100 (FIG. 51). Each individual lysingapparatus 5306 may include a respective disposable electric motorcoupled to drive the impeller. Each individual lysing apparatus 5306 mayinclude a first port 5308 a and a second port 5308 b (collectively 5308,only two called out in FIG. 53A). The ports 5308 may function as inletand/or outlets to a chamber (not called out in FIG. 53A or 53B).

As illustrated in FIG. 53B, the lysing manifold or array 5300 mayinclude a support structure 5310 to support one or more blocks or frames5302 and associated individual lysing apparatus 5306. In particular, thesupport structure 5310 may include rails 5310 a, 5310 b to hold theblock or frame 5302 and associated individual lysing apparatus 5306positioned relative to a structure that receives the lysed material, forexample a plate such as a micro-titer plate 5312. For instance, thesupport structure 5310 may hold the block or frame 5302 such that theassociated individual lysing apparatus 5306 are positioned aboverespective ones of a plurality of wells 5312 a (only one called out inFIG. 53B) of the micro-titer plate 5312. FIG. 53B shows only a singlelysing manifold or array 5300 carrying a single row of individual lysingapparatus 5306, constituting a one-dimensional array of lying apparatus5306. Alternatively, the support structure 5310 may carry additionallysing manifolds or arrays, each carrying a respective single row ofindividual lysing apparatus 5306. The individual lysing apparatus 5306carried by the plurality of lysing manifolds or arrays 5300 canconstitute a two-dimension array. As a further alternative, a singlelysing manifold or array 5300 may carry individual lysing apparatus 5306arranged in a two-dimensional array. As an even further alternatively, amotor and drive mechanism may be coupled to move a single lysingmanifold or array 5300 carrying the individual lysing apparatus 5306along the rails 5310 a, 5310 b of the support structure 5310. Thus, theone-dimensional array of lysing apparatus 5306 may be moved to address atwo-dimensional array of positions. Movement may be controlled manuallyor automatically, for example via one or more computer processors.

As described immediately above, individual lysing apparatus 5306 can bebundled together into a lysing manifold or array 5300 (e.g., one- or twodimensions) to facilitate multiplex processing. The distance betweencenters for these individual lysing apparatus 5306 can, for example, be9 mm or a multiple of 9 mm to match a standard format of a micro-titerplate 5312 (e.g., with 9 mm spacing, 96 well plate or greater).Similarly, the use of electric motors with diameters below 4.5 mm allowsthe manifold or array of lysing apparatus 5300 to be used formicro-titer plate formats with 4.5 mm spacing (e.g., 384 well plate).Bundling the individual lysing apparatus 5306 in strips or rows of 4, 8,6 or 12 may facilitate use for automated or semi-automated processing ofsamples in a micro-titer format. Additionally, if intake ports 5308 a ofthe individual lysing apparatus 5306 are designed to receive sample orspecimen from pipette tips, then the individual lysing apparatus 5306may be addressed by multichannel pipettors for either manual or roboticoperation. The block or frame 5302 may be fabricated monolithically froma single block of material that has been molded or cut-extruded withmultiple sites for the individual lysing apparatus 5306.

FIG. 54A and 54B show a cartridge style container 5400 configured toperform flow through lysis processing, according to one illustratedembodiment. In particular, FIG. 54A shows the container 5400 with oneend cap 5400 a removed to provide access to a chamber (not called out inFIG. 54A or 54B) formed by a body 5400 b of the container 5400, and theother end cap 5400 c fixed to the body 5400 b. FIG. 54B shows thecontainer 5400 with both end caps 5400 a, 5400 c fastened to the body5400 b of the container 5400. The cartridge style container 5404 may,for example, be employed with the oscillating arcuate motion basedapparatus (FIGS. 1-5), sometimes referred to herein as a bead beater.

The body 5400 b of the container 5400 may have openings 5400 d (only oneillustrated, in FIG. 54A) at opposed ends 5400 e, 5400 f thereof,providing access to the chamber formed by the body 5400 b of thecontainer 5400. These openings 5400 d may be relatively large toaccommodate samples or specimens in various states. As noted above, atleast one end cap 5400 a, 5400 b of the container 5400 is selectivelyremovable from the body 5400 b of the container 5400. Such providesaccess to the interior of the chamber for relatively large samples orspecimens. In some embodiments, both end caps 5400 a, 5400 c areselectively removable and fastenable to the body 5400 b of the container5400, while in other embodiments only one end cap 5400 a, 5400 c may beremovable. In such embodiments, the other end cap 5400 a, 5400 c may bea monolithic portion of the body 5400 b, or may be permanently securedthereto, for example via an adhesive, heat sealing or radio frequency(RF) welding.

Each of the end caps 5400 a, 5400 c may include a respective port 5402a, 5402 b (collectively 5402) that provides fluid communication to theinterior chamber of the body 5400 b from an exterior thereof. Such mayaccommodate flow through operation.

The cartridge style container 5400 and flow through lysing operation maybe used on virtually any cell type, for example plants, bacteria,spores, yeast, invertebrates and vertebrates. Additionally, there-closable end caps 5400 a, 5400 c advantageously allows the placementof a piece of sample tissue in the chamber, while maintaining flowthrough capability after the end cap 5400 a, 5400 c is fastened to closethe chamber. This may allow the lysing apparatus to function as ahomogenizer of tissues, for instance biopsy samples, mouse tail slices,leaf punches, seeds, etc. Such may eliminate the need to precede celllysis with a separate tissue homogenization act or step, which wouldotherwise typically require a separate piece of equipment.

A small disposable electric motor, such as that used to mix the beads inthe embodiment employing an impeller received in a chamber of beadblender (e.g., FIG. 16), may also be used for other integrated functionsrelated to analytical biochemistry. For example, such small disposableelectric motors may be used as part of, but not limited to, a pump, areversible pump, a valve, a mixer of reagents, a micro centrifuge, etc.Such disposable electric motors may even perform these functions incombination with performing other functions, such as, but not limitedto, lysing cells in the presence of lysing particulate or beads, whilepumping fluid (e.g., material to be lysed, lysed material, cleansers) atthe same time. For example, a pump may comprises an assay device withimpeller and a disposable electric motor, such as illustrated in FIG.16, with a check valve on either or both ports. The check valve(s)direct flow in one direction, while the fluid is driven by motion themotor imparts to the fluid via the impeller.

In both configurations (i.e., lysing apparatus with rapidly oscillatingarcuate motion sometimes referred to herein as bead beating or lysingapparatus with high angular velocity impeller sometimes referred toherein as bead blender), high energy is imparted to the fluid, in turncausing high velocities of lysing particulate or beads relative to eachother, which in turn causes high shear forces between lysing particulateor beads as they pass by relative to each other. Not to be limited bytheory, these shear forces are a possible explanation for surprisingultra rapid lysis of the cells. Other configurations that impart similarshear forces between the lysing particulate or beads may also providerapid cell lysing.

The flow through nature of some embodiments may allow for reuse of thesystem for processing additional samples or specimens. For example, theflow through nature may facilitate performance of one or more wash actsor steps to sterilize or otherwise sanitize or cleanse the system.Containers may be reused by cleaning and/or sterilizing the containerbetween uses. This may be coordinated with downstream processing of onesample or specimen such that the container may be made ready for anothersample or specimen during the downstream processing. One or more actsmay be employed to clean and/or sterilize the container, for exampleusing a high pH or low pH solution, bleach, detergent or combinationsthereof. Adjusting pH may advantageously reduce the number of wash actsor steps, since the pH can be easily neutralized. An alternativeapproach may be the use of di-ethyl-pyrocarbonate (DEPC). DEPC compoundcan destroy proteins and nucleic acid. This treatment may be followed bya single wash and then a flow of hot air. Because DEPC is so volatile,it may be removed by degradation and evaporation during the act ofpassing heated air over any surfaces treated with the DEPC.

Lysis efficiency or cell disruption appears to be affected by the ratioof particulate or bead volume to chamber volume. Higher efficiencyappears to occur when the volume of lysing particulate or beads isgreater than 50% of the volume of the chamber, with an upper limit. Notto be limited by theory, the assumption is that a denser population oflysing particulate or beads leads to a higher rate of collisions and/ora higher rate of proximal passes between lysing particulate or beadswith high shear force, thereby increasing the efficiency of lysis.Clearly, this advantage diminishes when the lysing particulate or beadsare packed too densely to move or are too dense to permit the electricmotor to function (e.g., over packed in bead blender apparatus). Intheory, the ratio of chamber volume to lysing particulate or bead volumefor both the oscillating arcuate motion based apparatus (i.e., beadbeater) and the rotational impeller based apparatus (i.e., bead blender)can be any number, but higher efficiencies will occur when the ratio isgreater than 1 to 1.

Lysis efficiency appears to be affected by a ratio of the volume of thelysis chamber to the volume of fluid in the lysis chamber. The highenergy methods of lysis of cells by mechanical means with lysisparticulate or beads such as by rapid oscillation of the chamber or fastrotation of a vane (i.e., impeller) are primarily designed to fill thelysis chamber entirely with fluid. It is possible to include a gap ofair in the chamber during lysis, however doing so will disadvantageouslyreduce lysis efficiency as the air gap is increased. This approach ofallowing an air gap tends to generate heat. However, the heat mayadvantageously be used to further denature components of the samplematrix or assist in elution of captured analyte. For example, in thecase of capture of DNA by sequence specific capture probes, the heatgenerated by lysing in the presence of an air gap or pocket may be usedto enhance the release and elution of DNA from the capture probes.

The various embodiments described above can be combined to providefurther embodiments. U.S. provisional patent application Ser. No.61/020,072 filed Jan. 9, 2008; International Patent Application SerialNo. PCT/US2009/030622 filed Jan. 9, 2009 and published as WO2009/089466; U.S. provisional patent application Ser. No. 61/117,012filed Nov. 21, 2008; U.S. provisional patent application Ser. No.61/220,984 filed Jun. 26, 2009; U.S. provisional patent application Ser.No. 61/317,604, filed Mar. 25, 2010; and U.S. non-provisionalapplication Ser. No. 12/732,070, filed Mar. 25, 2010 are incorporatedherein by reference, in their entirety. Aspects of the embodiments canbe modified, if necessary to employ concepts of the various patents,applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-110. (canceled)
 111. An article to perform flow-through lysis byoscillation on material to be lysed, the article comprising: at leastone wall forming at least one chamber having a first opening and atleast a second opening spaced from the first opening, the first and thesecond openings to provide fluid communication into the chamber from anexterior thereof; a particulate lysing material received in the chamber,the particulate material including a plurality of particles sized tolyse a material to be lysed and having a combined particulate volume; afirst filter received in the chamber between the first opening and theparticulate material, the first filter having a plurality of aperturessized to substantially pass the material to be lysed and to retain theparticulate material; a second filter received in the chamber betweenthe second opening and the particulate material, the second filterhaving a plurality of apertures sized to pass the material to be lysedand to retain the particulate material, wherein the first filter and thesecond filter form a particulate retainment area therebetween having avolume sufficiently greater than said combined particulate volume topermit the plurality of particles to move relative to one another toprovide shear forces when in use to perform lysis by oscillation onmaterial to be lysed; a first tube coupled to provide fluidcommunication to the first opening for material to be lysed; and asecond tube coupled to provide fluid communication from the secondopening for material that has been lysed; wherein the first tube and thesecond tube do not restrict the oscillation of the chamber and do notresonate in response to the oscillation.
 112. The article of claim 111,further comprising: an attachment structure proximate the first opening.113. The article of claim 111, further comprising: a first attachmentstructure to attach the first tube to the first opening; and a secondattachment structure to attach the second tube to the second opening.114. The article of claim 111, further comprising: a nipple about thefirst opening.
 115. The article of claim 111, further comprising: afirst nipple to attach the first tube about the first opening; and asecond nipple to attach the second tube about the second opening. 116.The article of claim 111 wherein the at least one wall is elongated andhas a first end and a second end opposed to the first end.
 117. Thearticle of claim 116 wherein the first opening is at the first end andthe second opening is at the second end.
 118. The article of claim 116wherein the at least one wall is cylindrically tubular.
 119. The articleof claim 111 wherein the particulate material is a plurality of beads.120. The article of claim 119 wherein the plurality of beads includes atleast one of ceramic beads, glass beads, zirconium beads, metal beads,plastic beads, and sand.
 121. The article of claim 119 wherein theplurality of beads have diameters in the range of approximately 100microns.
 122. The article of claim 119 wherein the plurality of beadshave diameters in the range of 50 microns to 150 microns.
 123. Thearticle of claim 111 wherein when in use a volume of the particulatematerial is greater than a volume of material to be lysed.
 124. Thearticle of claim 123 wherein when in use there is essentially no air inthe chamber.
 125. The article of claim 123 wherein the chamber has avolume that holds less than 60 μl of the material to be lysed.
 126. Thearticle of claim 123 wherein the chamber has a volume that holdsapproximately 10 μl to approximately 40 μl of the material to be lysed.127. The article of claim 111 wherein the first and the second filtersare fixed to the wall.